Case studies

Case 2.1 A child with muscle weakness

A 2-month-old child is admitted to hospital because his parents are concerned that he has been experiencing difficulty with feeding and becoming breathless. Examination reveals proximal muscle weakness. There is no clinical evidence that the peripheral nerves are involved. Routine blood investigations are normal. In particular, serum creatine kinase levels are normal (the enzyme is released from muscle fibres as they undergo cell death).

A biopsy of skeletal muscle shows excessive glycogen in muscle fibres, with large numbers of lysosomes also containing glycogen. Assay of muscle showed a greatly reduced level of acid alpha-glucosidase (GAA), a lysosomal enzyme. Subsequent echocardiogram shows abnormally thick cardiac muscles.

Q. Describe the functional and structural basis of this case. In particular, classify this type of disease within the spectrum of causes of disease. Why do you think the heart is found to be abnormal?

Case 2.1 Answer

Lysosomes are membrane-bound intracellular organelles and are part of the acid vesicle system. These contain hydrolases that operate at the acidic pH found within the lysosome. In the acid vesicle system there are more than 30 defined and specific acid hydrolases, which not only degrade abnormal large molecules but also recycle or process normal cell constituents. Lysosomes internalize material that is degraded by hydrolases. Among the many substances broken down within the lysosome is glycogen. The enzyme responsible for the breakdown of glycogen is GAA (formerly called acid maltase ).

In this case, GAA activity is greatly reduced in association with the accumulation of glycogen within muscle. This has led to muscle disease with clinical features of proximal muscle weakness and poor ability to feed.

This type of disease is called a lysosomal storage disorder and is caused by a genetic defect that leads to lack of activity of one of the enzymes normally found within lysosomes. The substance that would normally be broken down by the missing enzyme accumulates within the cells and causes damage. Clinical manifestations of lysosomal storage diseases are mostly related to damage to cells that are non-dividing permanent populations such as neurons, skeletal muscle and liver tissue.

The heart is abnormal because the cardiac muscle cells are also affected. Were an endomyocardial biopsy to be performed, this would also show abnormal accumulation of glycogen within cells.

Other causes of abnormal glycogen storage are also the result of genetic defects that lead to reduced activity of enzymes involved in glycogen metabolism.

This disease is defined as the following:

• A storage disorder – The disease is associated with abnormal accumulation and storage of a substance within cells.

• A lysosomal storage disease – The disease is caused by absence of a lysosomal enzyme.

• A glycogenosis – The disease is associated with the abnormal handling of glycogen.

Muscle diseases that are associated with abnormalities of skeletal muscle fibres are termed myopathies . In this case, the patient’s condition would be classified as a metabolic myopathy.

Cardiac diseases that are caused by primary defects in myocardial muscle fibres are termed cardiomyopathies . This child would be classed as having a cardiomyopathy.

Case 2.2 A tumour of unknown origin

A 37-year-old man has been referred to hospital for investigation. He had noted a swelling in his neck, and his doctor felt that this was because of enlarged lymph nodes. On examination, the man had several palpable enlarged lymph nodes in the right lower cervical region. Detailed imaging confirmed enlarged lymph nodes but no obvious associated lesions in other organs. A surgeon has removed an enlarged node and sent it for histological examination to determine the cause of disease. A poorly differentiated tumour is discovered but its origin is not clear from initial histology.

Q. How might immunohistochemical assessment using antibodies to different cell constituents help in diagnosis? Concentrate on how expression of intermediate filaments could help with the diagnosis. What other cellular markers might be considered?

Case 2.2 Answer

When a biopsy shows a tumour, optimal treatment depends on characterizing the type of tumour, which requires ascertaining its cell of origin. In most instances, this is not a diagnostic problem, and routine histological examination allows for a diagnosis to be made based on morphology alone. However, in some patients, histological examination using haemoxylin and eosin (H&E) staining does not allow the cell of origin of a tumour to be determined. The case under discussion is a common clinical problem: metastatic disease (i.e. a malignant tumour that has spread from its organ or tissue of origin to another site such as a lymph node or the liver) is discovered in the absence of any known primary site.

In such cases, standard histological examination is supplemented by immunohistochemical assessment of the tumour to detect markers that might point to the cell of origin. It is common to perform immunohistochemical staining using a panel of antibodies capable of detecting a wide range of possible tumours.

The intermediate filaments are composed of protein subunits and form part of the cytoskeleton. Different types of intermediate filaments characterize different cell types. The main types are as follows:

If immunostaining is performed using antibodies against specific intermediate filament proteins, it is possible to determine the cell of origin of a tumour.

In this case, expression of cytokeratins would lead to the conclusion that the tumour was of epithelial origin and was therefore some sort of carcinoma ( carcinoma is the term used to refer to malignant tumour of an epithelial tissue).

Epithelial cells from different tissues can also express specific proteins useful in diagnosis. For example:

• Tumours derived from prostatic epithelium may be detected by immunostaining for prostate-specific antigen or prostate-specific acid phosphatase.

• Tumours derived from the thyroid may be detected by immunostaining for thyroglobulin.

• Tumours derived from the breast may express oestrogen receptor.

• Tumours derived from the gastrointestinal tract may express carcinoembryonic antigen (CEA).

• Tumours of lymphoid tissues express markers of normal lymphoid cells.

• Tumours of muscle often express desmin, the intermediate filament of muscle.

It would be very unusual for a tumour of the nervous system to spread to lymph nodes. However, tumours that arise in the nervous system can be characterized by immunostaining for GFAP and neurofilament proteins to determine their cell of origin.

Any tumour of neuroendocrine type can be characterized by detecting proteins expressed on neurosecretory granules, such as synaptophysin or chromogranin A.

Vimentin is expressed by so many cell types that it is not particularly helpful in diagnosis.

In some patients, a tumour becomes so poorly differentiated that no diagnostically helpful pattern of expression of any protein is seen. Such anaplastic tumours generally carry a very poor prognosis.

Case 3.1 Nodules in the liver

A 62-year-old man is admitted to hospital with abdominal pain. On examination, he has an enlarged liver, and investigations show multiple nodules in the liver. Although a diagnosis of cancer spreading to the liver is strongly suspected, detailed imaging reveals no obvious site for a primary tumour. Under image guidance, a needle biopsy of one of the liver lesions is performed. This reveals unusual cells in the liver that are characterized by large, densely stained nuclei, variation in nuclear size and many mitoses.

Q. What do the histological features suggest as a likely diagnosis? What other histological assessments could be done to help refine the diagnosis? Concentrate on why these features do not fit with normal histology. What stains can be used to help determine cellular differentiation?

Case 3.1 Answer

Normal cells and tissues are characterized by a uniformity of cytology and a coherence of tissue architecture.

Cell division is usually restricted to a specific part of a tissue, for example, the basal layer of the epidermis of the skin or the crypts in the intestinal mucosa. Cells are usually of uniform size within any given tissue and tend not to show much variation in size or shape. In normal cells, the intensity of nuclear staining is related to the activity of the cell.

Neoplastic cells from malignant tumours are often found to have nuclei that are more darkly staining than in normal tissues (hyperchromatism). They also show variation in nuclear size and shape (pleomorphism), and evidence of cell proliferation exists in the form of mitoses.

In this case, the finding of cells with variable size and shape, mitoses and darkly stained nuclei suggests a neoplastic process and raises the possibility that the lesions in the liver are tumours.

In any tissue, a malignant process can be caused by a tumour arising locally (primary tumour) or spreading to the tissue from another site (metastatic tumour).

Histological assessment of a tumour is an important part of diagnosis and may be the investigation that suggests the original site of origin. This is important because prognosis and therapy of tumours are related to the site of origin and histogenesis.

Differentiation of tissues can be detected by conventional stains, immunohistochemical staining, electron microscopy and molecular genetic testing.

Conventional stains such as Alcian blue or periodic acid–Schiff (PAS) can detect the presence of mucin, which in this case would suggest that the tumour was epithelial in origin and of glandular differentiation; therefore the tumour would be classified as an adenocarcinoma.

Immunohistochemistry can be used to detect proteins expressed by cells that are specific to different cell types (see details as described in answer to Case 2.2). The majority of tumours that involve the liver are secondary, metastatic tumours that have spread from another site. Common sites of origin include carcinomas arising from the stomach or colon, lung and breast.

Primary malignant tumours may develop in the liver either arising from hepatocytes or from bile duct epithelium. In such cases markers can be applied using immunohistochemical staining and are related to the cell lineage of the tumour. As an example, a panel of antibodies might be used to investigate whether a tumour was a primary hepatocellular carcinoma derived from hepatocytes. Positive staining might be expected for cytokeratins 8 and 18, Hep Par1, glutamine synthetase, glypican-3, alpha-fetoprotein and CD10.

The key concept to take away from this case is that histological examination using a panel of antibodies using immunohistochemistry provides important clinical diagnostic information.

Case 4.1 Infant with a broken femur

Parents bring their 1-year-old daughter to the hospital as an emergency. They give a history that after a bath she had been pulling up to stand, when there was a ‘popping’ noise and she collapsed, screaming out in pain. She lay on the floor crying with pain. Her leg looked deformed and appeared broken.

An x-ray confirms a fractured femur. In the hospital, there was an initial suspicion of possible non-accidental injury. However, further detailed investigation showed that the child had a genetic abnormality in type I collagen formation of the type known to be associated with ‘brittle bones’.

Q. Explain how this molecular defect relates to abnormal bone fragility. Knowing that there are many types of collagen, each with a different gene and tissue distribution, predict which organs and tissues may be affected by genetically abnormal collagen.

Case 4.1 Answer

The strength of bone is determined by its extracellular matrix proteins and its mineralization. Osteoblasts elaborate the support matrix of bone, osteoid, which subsequently calcifies to form bone.

Osteoid is composed mainly of type I collagen, which is associated with the extracellular glycosaminoglycans (GAGs) chondroitin sulphate and keratin sulphate. Two glycoproteins, sialoprotein and osteocalcin, are mainly found in the bone matrix and bind calcium; therefore they may have a role in bone mineralization. In children, several types of so-called ‘brittle bone disease’ are recognized, properly called osteogenesis imperfecta (OI).

OI is a dominant inherited condition that leads to a disorder of type I collagen, which is the main extracellular matrix protein of bone. Several forms of OI have been described, and only the most common types (I–IV) are presented here.

Case 5.1 Sudden cardiac death

A 24-year-old male is brought into hospital by ambulance, having collapsed while running in a half-marathon. Paramedics had attended, and he was found to have no cardiac output and was in asystole. He was pronounced dead, and an autopsy was requested to establish the cause of death.

The pathologist found that the heart was greatly enlarged and showed hypertrophy of the left ventricle. The left ventricular wall was much thicker than normal. Histology showed that myocardial cells were greatly enlarged and had an abnormal pattern of myofibres. A diagnosis of hypertrophic cardiomyopathy was made. Further genetic testing showed a mutation in one of the genes coding for cardiac-specific myosin.

Q. Explain the relationship between the mutation in the gene and cardiac disease.

Case 5.1 Answer

Cardiac muscle is a form of striated muscle. Individual cardiac muscle cells are joined end to end at intercalated discs to form long fibres. Nuclei are typically in the centre of each cardiac muscle cell.

The term hypertrophic refers to the fact that the mass of the heart is enlarged by an increase in the size of individual cardiac muscle cells. The term cardiomyopathy implies that heart disease has resulted from a primary defect in the cardiac muscle.

The main contractile proteins in cardiac striated muscle are actin and myosin. Myosin interacts with several other proteins to form a regular array of contractile proteins arranged in striations.

The proteins involved in muscle contraction may be abnormal because of a mutation in the coding gene. In some patients with mutation in a gene form of cardiac myosin, contraction of muscle fibres is inefficient, leading to cardiac hypertrophy (enlargement of individual muscle cells that causes an increase in total mass of the myocardium).

It is now recognized that mutation in the genes coding for several muscle proteins, each of which is part of the contractile mechanism, can lead to hypertrophic cardiomyopathy. Mutations in the genes for cardiac myosin-binding protein C (MYBPC3) and β-myosin heavy chain (MYH7) account for about 80% of families with identified mutations.

Case 6.1 Possible epileptic seizure

A 52-year-old man is admitted to hospital because he has collapsed with a possible epileptic seizure. Clinical examination suggests that he has mild weakness of the right side of the body. A computed tomography (CT) scan shows an abnormal area in the left cerebral hemisphere, which is felt to be a possible brain tumour.

Q. What cells can give rise to tumours affecting the brain?

Case 6.1 Answer

The main cell types in the brain are neurons and glial cells comprising astrocytes, oligodendrocytes, ependymal cells and microglia. Outside the brain are epithelial cells associated with the meninges; such cells are called meningothelial cells . Tumours can arise from any of these cell types.

The most common primary tumours arise from glial cells (gliomas) in the form of astrocytic tumours. Less common are tumours of oligodendrocytes (oligodendrogliomas) and ependymal cells.

Tumours that contain neurons are not very common compared with those that are purely glial.

True tumours of the resting phagocytic cells of the brain, the microglia, are extremely rare. However, lymphoid cells that migrate through the brain can cause tumours – so-called cerebral lymphomas .

One of the most common tumours that affect the brain is derived from the epithelial cells of the meninges, which form meningiomas .

In childhood, tumours arise from small primitive cells that are believed to be related to the primitive neuroectoderm. Such tumours can have a variety of patterns of differentiation.

In adults, a common cause of a mass lesion (tumour) in the brain is metastasis of a malignant tumour arising from a primary site elsewhere in the body.

Magnetic resonance (MR) imaging often shows features which narrow the diagnosis to either a primary or metastatic tumour. Neurosurgical intervention and taking a biopsy of a tumour is usually required to get a histological diagnosis. As with other cases, immunohistochemical assessment of a biopsy is performed in order to characterize the histogenesis of any brain tumour. Increasingly, molecular assessment to determine genetic changes is also used to define the specific type of primary brain tumour and determine subsequent further treatment.

Case 6.2 Diabetes-related neuropathy

A 45-year-old patient with diabetes mellitus complains of numbness in the hands and feet. He has developed ulcers on the toes that are painless. Clinical examination shows reduced sensation to pain, touch and vibration in the hands and feet. A nerve conduction study shows a reduced speed of conduction (low-conduction velocity). A diagnosis of peripheral neuropathy is made.

Q. Describe the structural elements of peripheral nerve that may have become abnormal. Given that there is a reduced nerve conduction velocity, what component is most likely to be affected?

Case 6.2 Answer

A peripheral nerve is composed of the following:

• Axons

• Schwann cells, which make myelin

• Spindle-shaped fibroblast support cells, which produce fibrocollagenous tissue

• Blood vessels

There are three types of support tissue in a nerve trunk: the endoneurium, the perineurium and the epineurium.

Endoneurium is composed of longitudinally oriented collagen fibres, extracellular matrix material rich in glycosaminoglycans and sparse fibroblasts. It surrounds the individual axons and their associated Schwann cells, in addition to capillary blood vessels.

Perineurium surrounds groups of axons and endoneurium to form small bundles (fascicles). It is composed of seven or eight concentric layers of epithelium-like flattened cells separated by layers of collagen. The cells are joined by junctional complexes, and each layer of cells is surrounded by an external lamina.

Epineurium is an outer sheath of loose fibrocollagenous tissue that binds individual nerve fascicles into a nerve trunk. The epineurium may also include adipose tissue, along with a main muscular artery supplying the nerve trunk.

In a peripheral neuropathy, the structures most likely to be damaged are the axons or the myelin and Schwann cells. In some cases, disease is caused by abnormalities in blood vessels that lead to local ischaemia of a nerve.

The speed of conduction along nerves is limited by the electrical capacitance and resistance of the axon. Because wide axons have a lower capacitance than narrow ones, increasing the diameter of axons is a useful means of increasing the speed of nerve conduction. This is inefficient, however, because giant axons require high metabolic upkeep. The speed of conduction along axons is increased by myelination, with conduction taking place at the nodes of Ranvier. The abnormal reduction in conduction velocity seen in this patient suggests that the main problem is loss of myelin from nerves (demyelination).

Diabetic patients are especially prone to development of peripheral neuropathy.

Case 7.1 A man who was tired and weak

A 62-year-old man is admitted to hospital for investigation. He had gone to his family physician feeling generally unwell and tired. A full blood count had shown a greatly reduced red cell count, together with a reduction in the number of circulating white cells and platelets. Red cells were of normal size (normocytic) and contained normal amounts of haemoglobin (normochromic).

The neutrophil count was 0.5 × 10 ⁹ /L.

The platelet count was 20 × 10 ⁹ /L.

A diagnosis of pancytopenia was made, and further investigations were performed. A biopsy of the bone marrow was taken and showed a low cellularity affecting all precursors. A diagnosis of aplastic anaemia was made.

Q. Describe the structural and histological background to this case. Concentrate on describing the normal formation of the cells in the blood, and describe the functional complications that may be expected from disease.

Case 7.1 Answer

In aplastic anaemia, the quantity of all cellular elements in the blood is reduced because of failure of the bone marrow to manufacture cells.

In normal bone marrow, stem cells divide and produce committed progenitor cells that produce the various cell lineages.

All cellular elements of the blood originate from a common pluripotential progenitor stem cell (haemopoietic stem cell, HSC). These pluripotential stem cells are present in very small numbers at sites of blood cell formation, and even fewer of them can be found in the peripheral blood. Histologically, they resemble lymphocytes but can be identified by the use of immunohistochemical techniques because they have distinctive cell surface antigens. The pluripotential cells divide and give rise to cells with a more restricted line of growth.

It is possible to divide blood-forming cells into four groups, depending on capacity for self-renewal, cell division and ability to form different cell types. Pluripotential stem cells are capable of forming any type of blood cell; multipotential progenitor cells are capable of forming a specific but wide range of blood cells; committed progenitor cells are capable of forming only one or two types of blood cells; and maturing cells are undergoing structural differentiation to form one cell type and so are incapable of division.

Growth control of blood stem cells is through secreted growth factors and local cell contacts.

The best understood mechanism of control of growth of the different types of HSCs is through the action of growth factors. These substances are secreted systemically or locally and modulate three aspects of cell growth:

• Proliferation

• Differentiation

• Maturation

Less well understood in the control of blood cell formation is the role of local cell–cell contacts. Stromal cells in the bone marrow appear to be important in the control of differentiation and maturation; however, the signals involved are at present uncertain.

Aplastic anaemia can be associated with a variety of conditions and can be encountered as a disease of unknown cause:

• Reduction in platelets leads to increased tendency to bleed, with ease of bruising and bleeding from gums often being described by an affected person.

• Reduction in red cells leads to clinical features of anaemia, with tiredness, fatigue and breathlessness often being described by an affected person.

• Reduction in white cells leads to susceptibility to infection.

In this case, a bone marrow biopsy showed a lack of precursor cells for all formed elements in the marrow. This indicates that the reduced cellularity of all elements in the blood is caused by a failure of marrow production rather than a process of increased peripheral consumption or loss of cells.

Case 8.1 A boy with big lymph nodes

A 12-year-old boy is admitted to hospital for investigation of enlarged lymph nodes in the neck. He had been complaining of being unwell for about 6 weeks and had lost weight. He had what his mother described as drenching sweating attacks at night that often woke him up. On examination, he had palpable enlarged lymph nodes in the neck.

Surgical excision of a lymph node was performed so that it could be looked at histologically. The reporting pathologist noted the following: ‘There is expansion of germinal centres associated with extensive expansion of deep cortical areas. There are multiple aggregates of epithelioid macrophages with multinucleate forms within the lymph node. These are surrounded by zones of activated T cells. The appearances are those of granulomatous inflammation. In the centre of many granulomas are areas of necrosis. These features suggest that tuberculous infection is the most likely cause’.

Q. Describe the anatomical and histological background to the case. Concentrate on describing the normal structure of a lymph node.

Case 8.1 Answer

This clinical case illustrates a common problem: an enlarged lymph node of uncertain cause. The lymph node is a bean-shaped organ with a fibrocollagenous capsule from which fibrous trabeculae extend into the node to form a supporting framework.

Lymph nodes contain three functional compartments:

• A network of endothelial-lined lymphatic sinuses continuous with the lumina of the afferent and efferent lymphatic vessels

• A network of small blood vessels, where circulating lymphocytes enter the node

• A parenchymal compartment composed of superficial cortex, deep cortex and medulla

The superficial cortex of the lymph node contains the densely staining spheroid aggregations of lymphocytes (lymphoid follicles). Some of the follicles (primary follicles) are of fairly uniform staining density; however, most of the follicles that respond to antigen have less densely staining germinal centres and are described as secondary follicles. The lymphocyte population of the follicles consists predominantly of B cells, but there are smaller populations of TH cells, macrophages and accessory cells.

The cell population of the deep cortex consists of lymphocytes and accessory cells, which constantly move in and out of the region. T cells dominate the deep cortex, entering the node from the blood via the high endothelial venules (HEVs) and leaving via the efferent lymphatics. When activated, T cells enlarge to form lymphoblasts. These then proliferate to produce an expanded clone of activated T cells. In a T-cell–dominated immunological response, the deep cortex may expand into the medulla, producing a so-called ‘paracortical reaction’. Activated T cells are then disseminated via the circulation to peripheral sites, where much of their activity occurs.

In this case, the lymph node is characterized by expansion of both cortex (B-cell) and deep cortical (T-cell) areas. One aspect of the function of activated T cells is the secretion of cytokines and the recruitment and activation of macrophages. This is the functional background to the formation of aggregates of histiocytic cells to form structures called granulomas .

Several disorders trigger T cells to activate macrophages and cause a granulomatous inflammatory response. Histological features of the granuloma can point to a likely cause. When associated with necrosis, granulomatous inflammation in a lymph node is likely to have been caused by tuberculous infection. Further investigation to characterize the presence of mycobacteria would normally be performed.